The broad objectives of the Parent Proposal are to understand the mechanism of microtubule dynamics in vivo and the relationship between this dynamic activity and important cell biological functions including organization of the cytoplasm, the generation of cell form, cell division, and cell locomotion. The conceptual framework underlying the Parent Proposal is that the organization of microtubules and their turnover in cells result from an interplay of cellular components. Two important classes of component are: (1) the centrosome as a nucleating and anchoring structure and (2) cytoplasmic factors that effect the dynamic activity of the minus and plus ends, respectively. Of particular focus in the Parent Proposal will be a novel, recently formulated hypothesis for microtubule turnover which they call the "minus-end pathway." In this application, the investigators propose to explore a related issue. A paradox in the microtubule dynamics field is the apparent contradiction between rapid microtubule turnover and tempered dynamic instability of plus ends. Experimental analysis and theoretical calculations indicate that tempered dynamic instability can not account for the rapid formation and turnover of microtubules tens of micrometers long commonly observed in a variety of cells. The investigators suggest that this discrepancy results from an incomplete understanding of microtubule behavior resulting from observations being limited to the cell periphery while behavior in the internal cytoplasm remains essentially unknown. The specific hypothesis to be tested is the "persistent growth" model for microtubule formation and turnover. According to this view, microtubules released from the centrosome depolymerize from their minus end and are replaced by microtubules nucleated at the centrosome which grow by continuous elongation towards the cell margin. Only near the cell margin do microtubules display the random stochastic behavior termed dynamic instability. Novel methods employing digital fluorescence microscopy will be developed to obtain data on microtubule behavior in the cell interior.